Cost-effective 3D lung tissue spheroid as a model for SARS-CoV-2 infection and drug screening.

BACKGROUND: The SARS-CoV-2 pandemic has spurred an unparalleled scientific endeavor to elucidate the virus' structure, infection mechanisms, and pathogenesis. Two-dimensional culture systems have been instrumental in shedding light on numerous aspects of COVID-19. However, these in vitro systems lack the physiological complexity to comprehend the infection process and explore treatment options. Three-dimensional (3D) models have been proposed to fill the gap between 2D cultures and in vivo studies. Specifically, spheroids, composed of lung cell types, have been suggested for studying SARS-CoV-2 infection and serving as a drug screening platform. METHODS: 3D lung spheroids were prepared by coculturing human alveolar or bronchial epithelial cells with human lung stromal cells. The morphology, size, and ultrastructure of spheroids before and after SARS-CoV-2 infection were analyzed using optical and electron microscopy. Immunohistochemistry was used to detect spike protein and, thus, the virus presence in the spheroids. Multiplex analysis elucidated the cytokine release after virus infection. RESULTS: The spheroids were stable and kept their size and morphology after SARS-CoV-2 infection despite the presence of multivesicular bodies, endoplasmic reticulum rearrangement, tubular compartment-enclosed vesicles, and the accumulation of viral particles. The spheroid responded to the infection releasing IL-6 and IL-8 cytokines. CONCLUSION: This study demonstrates that coculture spheroids of epithelial and stromal cells can serve as a cost-effective infection model for the SARS-CoV-2 virus. We suggest using this 3D spheroid as a drug screening platform to explore new treatments related to the cytokines released during virus infection, especially for long COVID treatment.

Effect of Polyplex Size on Penetration into Tumor Spheroids.

Ovarian cancer is one of the most lethal gynecological cancers in the world. In recent years, nucleic acid (NA)-based formulations have been shown to be promising treatments for ovarian cancer, including tumor nodules. However, gene therapy is not that far advanced in clinical reality due to unfavorable physicochemical properties of the NAs, such as high molecular weight, poor cellular uptake, rapid degradation by nucleases, etc. One of the strategies used to overcome these drawbacks is the complexation of anionic NAs via electrostatic interactions with cationic polymers, resulting in the formation of so-called polyplexes. In this work, the role of the size of pDNA and siRNA polyplexes on their penetration into ovarian-cancer-based tumor spheroids was investigated. For this, a methoxypoly(ethylene glycol) poly(2-(dimethylamino)ethyl methacrylate) (mPEG-pDMAEMA) diblock copolymer was synthesized as a polymeric carrier for NA binding and condensation with either plasmid DNA (pDNA) or short interfering RNA (siRNA). When prepared in HEPES buffer (10 mM, pH 7.4) at a nitrogen/phosphate (N/P) charge ratio of 5 and pDNA polyplexes were formed with a size of 162 ± 11 nm, while siRNA-based polyplexes displayed a size of 25 ± 2 nm. The polyplexes had a slightly positive zeta potential of +7-8 mV in the same buffer. SiRNA and pDNA polyplexes were tracked in vitro into tumor spheroids, resembling in vivo avascular ovarian tumor nodules. For this purpose, reproducible spheroids were obtained by coculturing ovarian carcinoma cells with primary mouse embryonic fibroblasts in different ratios (5:2, 1:1, and 2:5). Penetration studies revealed that after 24 h of incubation, siRNA polyplexes were able to penetrate deeper into the homospheroids (composed of only cancer cells) and heterospheroids (cancer cells cocultured with fibroblasts) compared to pDNA polyplexes which were mainly located in the rim. The penetration of the polyplexes was slowed when increasing the fraction of fibroblasts present in the spheroids. Furthermore, in the presence of serum siRNA polyplexes encoding for luciferase showed a high cellular uptake in 2D cells resulting in ∼50% silencing of luciferase expression. Taken together, these findings show that self-assembled small siRNA polyplexes have good potential as a platform to test ovarian tumor nodulus penetration..

Novel Correlation between TGF-ß1/-ß3 and Hormone Receptors in the Human Corneal Stroma.

This study investigated the interplay between transforming growth factor beta (TGF-ß1/T1 and TGF-ß3/T3), and sex hormone receptors using our 3D in vitro cornea stroma model. Primary human corneal fibroblasts (HCFs) from healthy donors were plated in transwells at 106 cells/well and cultured for four weeks. HCFs were supplemented with stable vitamin C (VitC) and stimulated with T1 or T3. 3D construct proteins were analyzed for the androgen receptor (AR), progesterone receptor (PR), estrogen receptor alpha (ERα) and beta (ERß), luteinizing hormone receptor (LHR), follicle-stimulating hormone receptor (FSHR), gonadotropin-releasing hormone receptor (GnRHR), KiSS1-derived peptide receptor (KiSS1R/GPR54), and follicle-stimulating hormone subunit beta (FSH-B). In female constructs, T1 significantly upregulated AR, PR, ERα, FSHR, GnRHR, and KiSS1R. In male constructs, T1 significantly downregulated FSHR and FSH-B and significantly upregulated ERα, ERß, and GnRHR. T3 caused significant upregulation in expressions PR, ERα, ERß, LHR, FSHR, and GNRHR in female constructs, and significant downregulation of AR, ERα, and FSHR in male constructs. Semi-quantitative Western blot findings present the interplay between sex hormone receptors and TGF-ß isoforms in the corneal stroma, which is influenced by sex as a biological variable (SABV). Additional studies are warranted to fully delineate their interactions and signaling mechanisms.

Neurodevelopmental toxicity assessment of flame retardants using a human DNT in vitro testing battery.

Due to their neurodevelopmental toxicity, flame retardants (FRs) like polybrominated diphenyl ethers are banned from the market and replaced by alternative FRs, like organophosphorus FRs, that have mostly unknown toxicological profiles. To study their neurodevelopmental toxicity, we evaluated the hazard of several FRs including phased-out polybrominated FRs and organophosphorus FRs: 2,2',4,4'-tetrabromodiphenylether (BDE-47), 2,2',4,4',5-pentabromodiphenylether (BDE-99), tetrabromobisphenol A, triphenyl phosphate, tris(2-butoxyethyl) phosphate and its metabolite bis-(2-butoxyethyl) phosphate, isodecyl diphenyl phosphate, triphenyl isopropylated phosphate, tricresyl phosphate, tris(1,3-dichloro-2-propyl) phosphate, tert-butylphenyl diphenyl phosphate, 2-ethylhexyl diphenyl phosphate, tris(1-chloroisopropyl) phosphate, and tris(2-chloroethyl) phosphate. Therefore, we used a human cell-based developmental neurotoxicity (DNT) in vitro battery covering a large variety of neurodevelopmental endpoints. Potency according to the respective most sensitive benchmark concentration (BMC) across the battery ranked from <1 µM (5 FRs), 1<10 µM (7 FRs) to the >10 µM range (3 FRs). Evaluation of the data with the ToxPi tool revealed a distinct ranking (a) than with the BMC and (b) compared to the ToxCast data, suggesting that DNT hazard of these FRs is not well predicted by ToxCast assays. Extrapolating the DNT in vitro battery BMCs to human FR exposure via breast milk suggests low risk for individual compounds. However, it raises a potential concern for real-life mixture exposure, especially when different compounds converge through diverse modes-of-action on common endpoints, like oligodendrocyte differentiation in this study. This case study using FRs suggests that human cell-based DNT in vitro battery is a promising approach for neurodevelopmental hazard assessment and compound prioritization in risk assessment.

The impact of impaired DNA mobility on gene electrotransfer efficiency: analysis in 3D model.

BACKGROUND: Gene electrotransfer is an established method that enables transfer of DNA into cells with electric pulses. Several studies analyzed and optimized different parameters of gene electrotransfer, however, one of main obstacles toward efficient electrotransfection in vivo is relatively poor DNA mobility in tissues. Our aim was to analyze the effect of impaired mobility on gene electrotransfer efficiency experimentally and theoretically. We applied electric pulses with different durations on plated cells, cells grown on collagen layer and cells embedded in collagen gel (3D model) and analyzed gene electrotransfer efficiency. In order to analyze the effect of impaired mobility on gene electrotransfer efficiency, we applied electric pulses with different durations on plated cells, cells grown on collagen layer and cells embedded in collagen gel (3D model) and analyzed gene electrotransfer efficiency. RESULTS: We obtained the highest transfection in plated cells, while transfection efficiency of embedded cells in 3D model was lowest, similarly as in in vivo. To further analyze DNA diffusion in 3D model, we applied DNA on top or injected it into 3D model and showed, that for the former gene electrotransfer efficiency was similarly as in in vivo. The experimental results are explained with theoretical analysis of DNA diffusion and electromobility. CONCLUSION: We show, empirically and theoretically that DNA has impaired electromobility and especially diffusion in collagen environment, where the latter crucially limits electrotransfection. Our model enables optimization of gene electrotransfer in in vitro conditions.

Organophosphorus flame retardants are developmental neurotoxicants in a rat primary brainsphere in vitro model.

Due to regulatory bans and voluntary substitutions, halogenated polybrominated diphenyl ether (PBDE) flame retardants (FR) are increasingly substituted by mainly organophosphorus FR (OPFR). Leveraging a 3D rat primary neural organotypic in vitro model (rat brainsphere), we compare developmental neurotoxic effects of BDE-47-the most abundant PBDE congener-with four OPFR (isopropylated phenyl phosphate-IPP, triphenyl phosphate-TPHP, isodecyl diphenyl phosphate-IDDP, and tricresyl phosphate (also known as trimethyl phenyl phosphate)-TMPP). Employing mass spectroscopy-based metabolomics and transcriptomics, we observe at similar human-relevant non-cytotoxic concentrations (0.1-5 µM) stronger developmental neurotoxic effects by OPFR. This includes toxicity to neurons in the low µM range; all FR decrease the neurotransmitters glutamate and GABA (except BDE-47 and TPHP). Furthermore, n-acetyl aspartate (NAA), considered a neurologic diagnostic molecule, was decreased by all OPFR. At similar concentrations, the FR currently in use decreased plasma membrane dopamine active transporter expression, while BDE-47 did not. Several findings suggest astrogliosis induced by the OPFR, but not BDE-47. At the 5 µM concentrations, the OPFR more than BDE-47 interfered with myelination. An increase of cytokine gene and receptor expressions suggests that exposure to OPFR may induce an inflammatory response. Pathway/category overrepresentation shows disruption in 1) transmission of action potentials, cell-cell signaling, synaptic transmission, receptor signaling, (2) immune response, inflammation, defense response, (3) cell cycle and (4) lipids metabolism and transportation. Taken together, this appears to be a case of regretful substitution with substances not less developmentally neurotoxic in a primary rat 3D model.

Evaluation of the toxicity of sodium dodecyl sulphate (SDS) in the MucilAir™ human airway model in vitro.

The aim of the study was to use multiple in vitro assays to assess the effects of a model irritant, sodium dodecyl sulphate (SDS) (≤10 mM (0.29 %, w/v)), on an in vitro model of the airway, MucilAir™. The use of MucilAir™ in recovery studies was also explored. A 24 h exposure increased IL-8 release at an SDS concentration ≥0.63 mM (0.018 %, w/v). Mucin secretion increased and transepithelial electrical resistance (TEER) decreased at SDS concentrations ≥1.25 mM (0.04 %, w/v). Cytotoxicity (lactate dehydrogenase (LDH) release into basolateral chamber) was observed at SDS concentrations of ≥2.5 mM (0.07 %, w/v). The sensitivity of the assays was IL-8 release > TEER = mucin secretion > LDH release. After 7 days, full or partial recovery was observed for intermediate concentrations of SDS using all assays but not at 5 and 10 mM SDS. Morphologically, erosion and cell loss were observed at these concentrations. Resazurin metabolism at 7 days tended to decrease in a dose-dependent manner at SDS concentrations above 2.5 mM (0.07 %, w/v). Together, these data support a No Observable Effect Level of 0.31 mM (0.009 % w/v) SDS and the use of MucilAir™ as a relevant model for airway toxicity studies.

Identification of miR-199a-5p, miR-214-3p and miR-99b-5p as Fibrosis-Specific Extracellular Biomarkers and Promoters of HSC Activation.

Liver fibrosis is characterized by the accumulation of extracellular matrix (ECM) resulting in the formation of fibrous scars. In the clinic, liver biopsies are the standard diagnostic method despite the potential for clinical complications. miRNAs are single-stranded, non-coding RNAs that can be detected in tissues, body fluids and cultured cells. The regulation of many miRNAs has been linked to tissue damage, including liver fibrosis in patients, resulting in aberrant miRNA expression/release. Experimental evidence also suggests that miRNAs are regulated in a similar manner in vitro and could thus serve as translational in vitro-in vivo biomarkers. In this work, we set out to identify and characterize biomarkers for liver fibrosis that could be used in vitro and clinically for research and diagnostic purposes. We focused on miRNAs released from hepatic 3D cultures exposed to methotrexate (MTX), which causes fibrosis, and acetaminophen (APAP), an acute hepatotoxicant with no clinically relevant association to liver fibrosis. Using a 3D in vitro model, we corroborated compound-specific responses as we show MTX induced a fibrotic response, and APAP did not. Performing miRNA-seq of cell culture supernatants, we identified potential miRNA biomarkers (miR-199a-5p, miR-214-3p, niRNA-125a-5p and miR-99b-5p) that were associated with a fibrotic phenotype and not with hepatocellular damage alone. Moreover, transfection of HSC with miR-199a-5p led to decreased expression of caveolin-1 and increased α-SMA expression, suggesting its role in HSC activation. In conclusion, we propose that extracellular miR-214-3p, miR-99b-5p, miR-125a-5p and specifically miR-199a-5p could contribute towards a panel of miRNAs for identifying liver fibrosis and that miR-199a-5p, miR-214-3p and miR-99b-5p are promoters of HSC activation.

Interstitial flow regulates in vitro three-dimensional self-organized brain micro-vessels.

Cell culture under medium flow has been shown to favor human brain microvascular endothelial cells function and maturation. Here a three-dimensional in vitro model of the human brain microvasculature, comprising brain microvascular endothelial cells but also astrocytes, pericytes and a collagen type I microfiber - fibrin based matrix, was cultured under continuous medium flow in a pressure driven microphysiological system (10 kPa, in 60-30 s cycles). The cells self-organized in micro-vessels perpendicular to the shear flow. Comparison with static culture showed that the resulting interstitial flow enhanced a more defined micro-vasculature network, with slightly more numerous lumens, and a higher expression of transporters, carriers and tight junction genes and proteins, essential to the blood-brain barrier functions.

Integrated mRNA and microRNA transcriptome profiling during differentiation of human nasal polyp epithelium reveals an altered ciliogenesis.

BACKGROUND: Human adult basal stem/progenitor cells (BSCs) obtained from chronic rhinosinusitis with nasal polyps (CRSwNP) when differentiated in an air-liquid interface (ALI) usually provide a pseudostratified airway epithelium with similar abnormalities than original in vivo phenotype. However, the intrinsic mechanisms regulating this complex process are not well defined and their understanding could offer potential new therapies for CRSwNP (incurable disease). METHODS: We performed a transcriptome-wide analysis during in vitro mucociliary differentiation of human adult BSCs from CRSwNP, compared to those isolated from control nasal mucosa (control-NM), in order to identify which key mRNA and microRNAs are regulating this complex process in pathological and healthy conditions. RESULTS: A number of genes, miRs, biological processes, and pathways were identified during mucociliary differentiation of both CRSwNP and control-NM epithelia, and notably, we have demonstrated for the first time that genetic transcriptional program responsible of ciliogenesis and cilia function is significantly impaired in CRSwNP epithelium, presumably produced by an altered expression of microRNAs, particularly of those miRs belonging to mir-34 and mi-449 families. CONCLUSIONS: This study provides for the first time a novel insight into the molecular basis of sinonasal mucociliary differentiation, demonstrating that transcriptome related to ciliogenesis and cilia function is significantly impaired during differentiation of CRSwNP epithelium due to an altered expression of microRNAs.

ALI multilayered co-cultures mimic biochemical mechanisms of the cancer cell-fibroblast cross-talk involved in NSCLC MultiDrug Resistance.

BACKGROUND: Lung cancer is the leading cause of cancer-related deaths worldwide. This study focuses on its most common form, Non-Small-Cell Lung Cancer (NSCLC). No cure exists for advanced NSCLC, and patient prognosis is extremely poor. Efforts are currently being made to develop effective inhaled NSCLC therapies. However, at present, reliable preclinical models to support the development of inhaled anti-cancer drugs do not exist. This is due to the oversimplified nature of currently available in vitro models, and the significant interspecies differences between animals and humans. METHODS: We have recently established 3D Multilayered Cell Cultures (MCCs) of human NSCLC (A549) cells grown at the Air-Liquid Interface (ALI) as the first in vitro tool for screening the efficacy of inhaled anti-cancer drugs. Here, we present an improved in vitro model formed by growing A549 cells and human fibroblasts (MRC-5 cell line) as an ALI multilayered co-culture. The model was characterized over 14-day growth and tested for its response to four benchmarking chemotherapeutics. RESULTS: ALI multilayered co-cultures showed an increased resistance to the four drugs tested as compared to ALI multilayered mono-cultures. The signalling pathways involved in the culture MultiDrug Resistance (MDR) were influenced by the cancer cell-fibroblast cross-talk, which was mediated through TGF-ß1 release and subsequent activation of the PI3K/AKT/mTOR pathway. As per in vivo conditions, when inhibiting mTOR phosphorylation, MDR was triggered by activation of the MEK/ERK pathway activation and up-regulation in cIAP-1/2 expression. CONCLUSIONS: Our study opens new research avenues for the development of alternatives to animal-based inhalation studies, impacting the development of anti-NSCLC drugs.

A novel spheroid-based co-culture model mimics loss of keratinocyte differentiation, melanoma cell invasion, and drug-induced selection of ABCB5-expressing cells.

BACKGROUND: Different 3D-cell culture approaches with varying degrees of complexity have been developed to serve as melanoma models for drug testing or mechanistic studies. While these 3D-culture initiatives are already often superior to classical 2D approaches, they are either composed of only melanoma cells or they are so complex that the behavior of individual cell types is hard to understand, and often they are difficult to establish and expensive. METHODS: This study used low-attachment based generation of spheroids composed of up to three cell types. Characterization of cells and spheroids involved cryosectioning, immunofluorescence, FACS, and quantitative analyses. Statistical evaluation used one-way ANOVA with post-hoc Tukey test or Student's t-test. RESULTS: The tri-culture model allowed to track cellular behavior in a cell-type specific manner and recapitulated different characteristics of early melanoma stages. Cells arranged into a collagen-IV rich fibroblast core, a ring of keratinocytes, and groups of highly proliferating melanoma cells on the outside. Regularly, some melanoma cells were also found to invade the fibroblast core. In the absence of melanoma cells, the keratinocyte ring stratified into central basal-like and peripheral, more differentiated cells. Conversely, keratinocyte differentiation was clearly reduced upon addition of melanoma cells. Treatment with the cytostatic drug, docetaxel, restored keratinocyte differentiation and induced apoptosis of external melanoma cells. Remaining intact external melanoma cells showed a significantly increased amount of ABCB5-immunoreactivity. CONCLUSIONS: In the present work, a novel, simple spheroid-based melanoma tri-culture model composed of fibroblasts, keratinocytes, and melanoma cells was described. This model mimicked features observed in early melanoma stages, including loss of keratinocyte differentiation, melanoma cell invasion, and drug-induced increase of ABCB5 expression in external melanoma cells.

A multi-site metastasis-on-a-chip microphysiological system for assessing metastatic preference of cancer cells.

Metastatic disease remains one of the primary reasons for cancer-related deaths, yet the majority of in vitro cancer models focus on the primary tumor sites. Here, we describe a metastasis-on-a-chip device that houses multiple bioengineered three-dimensional (3D) organoids, established by a 3D photopatterning technique employing extracellular matrix-derived hydrogel biomaterials. Specifically, cancer cells begin in colorectal cancer (CRC) organoid, which resides in a single microfluidic chamber connected to multiple downstream chambers in which liver, lung, and endothelial constructs are housed. Under recirculating fluid flow, tumor cells grow in the primary site, eventually enter circulation, and can be tracked via fluorescent imaging. Importantly, we describe that in the current version of this platform, HCT116 CRC cells preferentially home to the liver and lung constructs; the corresponding organs of which CRC metastases arise the most in human patients. We believe that in subsequent studies this platform can be implemented to better understand the mechanisms underlying metastasis, perhaps resulting in the identification of targets for intervention.

Desmoglein 3 - Influence on oral carcinoma cell migration and invasion.

Desmoglein 3 (Dsg3) is an adhesion receptor in desmosomes, but its role in carcinoma cell migration and invasion is mostly unknown. Our aim was to quantitatively analyse the motion of Dsg3-modified carcinoma cells in 2D settings and in 3D within tumour microenvironment mimicking (TMEM) matrices. We tested mutant constructs of C-terminally truncated Dsg3 (∆238 and ∆560), overexpressed full-length (FL) Dsg3, and empty vector control (Ct) of buccal mucosa squamous cell carcinoma (SqCC/Y1) cells. We captured live cell images and analysed migration velocities and accumulated and Euclidean distances. We compared rodent collagen and Matrigel® with human Myogel TMEM matrices for these parameters in 3D sandwich, in which we also tested the effects of monoclonal antibody AK23, which targets the EC1 domain of Dsg3. In monolayer culture, FL and both truncated constructs migrated faster and had higher accumulated distances than Ct cells. However, in the 3D assays, only the mutants invaded faster relative to Ct cells. Of the mutants, the shorter form (Δ238) exhibited faster migration and invasion than Δ560 cells. In the Transwell, all of the cells invaded faster through Myogel than Matrigel® coated wells. In 3D sandwich, AK23 antibody inhibited only the invasion of FL cells. We conclude that different experimental 2D and 3D settings can markedly influence the movement of oral carcinoma cells with various Dsg3 modifications.

Toxicity of ionizing radiation (IR) in a human induced pluripotent stem cell (hiPSC)-derived 3D early neurodevelopmental model.

Prenatal brain development is a complex and sensitive process, highly susceptible to environmental influences such as pollutants, stress, malnutrition, drugs, tobacco exposure, or ionizing radiation (IR). Disturbances in development may cause life-long disabilities and diseases, such as ADHD, childhood cancers, cognitive problems, depression, anxiety and more severe developmental disabilities. Due to increasing medical imaging, radiation therapy, natural terrestrial radiation, radioactive pollution and long-distance flights, humans are increasingly exposed to IR. However, data on impact of IR on very early human brain development are scarce, particularly in the very first weeks of gestation. Here we investigated the effects of low-dose X-ray IR (1 Gy) in a 3D early brain developmental model derived from human pluripotent stem cells. In this model very early neural stem cells, neuroectodermal progenitor cells (NEP), were exposed to low-dose IR and direct as well as delayed effects were investigated. Expression of 20 different marker genes crucial for normal neural development was determined 48 h and 9 days post IR (pIR). All but one of the analyzed marker genes were reduced 48 h after IR, and all but seven genes normalized their expression by day 9 pIR. Among the seven markers were genes involved in neurodevelopmental and growth abnormalities. Moreover, we could show that stemness of the NEP was reduced after IR. We were thus able to identify a significant impact of radiation in cells surviving low-dose IR, suggesting that low-dose IR could have a negative impact on the early developing human brain, with potential later detrimental effects.

3D Engineering of Ocular Tissues for Disease Modeling and Drug Testing.

The success rate from investigational new drug filing to drug approval has remained low for decades despite major scientific and technological advances, and a steady increase of funding and investment. The failure to demonstrate drug efficacy has been the major reason that drug development does not progress beyond phase II and III clinical trials. The combination of two-dimensional (2D) cellular in vitro and animal models has been the gold standard for basic science research and preclinical drug development studies. However, most findings from these systems fail to translate into human trials because these models only partly recapitulate human physiology and pathology. The lack of a dynamic three-dimensional microenvironment in 2D cellular models reduces the physiological relevance, and for these reasons, 3D and microfluidic model systems are now being developed as more native-like biological assay platforms. 3D cellular in vitro systems, microfluidics, self-organized organoids, and 3D biofabrication are the most promising technologies to mimic human physiology because they provide mechanical cues and a 3D microenvironment to the multicellular components. With the advent of human-induced pluripotent stem cell (iPSC) technology, the 3D dynamic in vitro systems further enable extensive access to human-like tissue models. As increasingly complex 3D cellular systems are produced, the use of current visualization technologies is limited due to the thickness and opaqueness of 3D tissues. Tissue-clearing techniques improve light penetration deep into tissues by matching refractive indices among the 3D components. 3D segmentation enables quantitative measurements based on 3D tissue images. Using these state-of-the-art technologies, high-throughput screening (HTS) of thousands of drug compounds in 3D tissue models is slowly becoming a reality. In order to screen thousands of compounds, machine learning will need to be applied to help maximize outcomes from the use of cheminformatics and phenotypic approaches to drug screening. In this chapter, we discuss the current 3D ocular models recapitulating physiology and pathology of the back of the eye and further discuss visualization and quantification techniques that can be implemented for drug screening in ocular diseases.

Unravelling the stromal-nerve interactions in the human diabetic cornea.

Corneal defects due to diabetes mellitus (DM) may cause severe vision impairments. Current studies focus on the corneal epithelium and nerve defects neglecting the corneal stroma. The aim of this study was to develop a 3D in vitro model to examine the interactions between corneal stroma and nerves in the context of DM. Primary human corneal stromal fibroblasts isolated from healthy (HCFs), Type 1 (T1DM) and Type 2 (T2DM) patients were stimulated with stable ascorbic acid to secrete and assemble an extracellular matrix (ECM). Human neuronal cells were then seeded on top and differentiated to create the 3D co-cultures. Our data revealed successful co-culture of stromal fibroblasts and neuronal cells with large elongated neuron extensions. T2DM showed significant upregulation of Collagen III and IGF1 when compared to T1DM. Interestingly, upon nerve addition, those markers returned to HCF levels. Neuronal markers were also differentially modulated with T2DM co-cultures expressing high levels of ßIII tubulin where T1DM co-cultures expressed Substance P. . Overall, our unique 3D co-culture model provides us with a tool that can be utilized for both molecular and therapeutic studies for diabetic keratopathy.

Vascularized Cardiac Spheroids as Novel 3D in vitro Models to Study Cardiac Fibrosis.

Spheroid cultures are among the most explored cellular biomaterials used in cardiovascular research, due to their improved integration of biochemical and physiological features of the heart in a defined architectural three-dimensional microenvironment when compared to monolayer cultures. To further explore the potential use of spheroid cultures for research, we engineered a novel in vitro model of the heart with vascularized cardiac spheroids (VCSs), by coculturing cardiac myocytes, endothelial cells, and fibroblasts isolated from dissociated rat neonatal hearts (aged 1-3 days) in hanging drop cultures. To evaluate the validity of VCSs in recapitulating pathophysiological processes typical of the in vivo heart, such as cardiac fibrosis, we then treated VCSs with transforming growth factor beta 1 (TGFß1), a known profibrotic agent. Our mRNA analysis demonstrated that TGFß1-treated VCSs present elevated levels of expression of connective tissue growth factor, fibronectin, and TGFß1 when compared to control cultures. We demonstrated a dramatic increase in collagen deposition following TGFß1 treatment in VCSs in the PicroSirius Red-stained sections. Doxorubicin, a renowned cardiotoxic and profibrotic agent, triggered apoptosis and disrupted vascular networks in VCSs. Taken together, our findings demonstrate that VCSs are a valid model for the study of the mechanisms involved in cardiac fibrosis, with the potential to be used to investigate novel mechanisms and therapeutics for treating and preventing cardiac fibrosis in vitro.

Cytochrome P450 induction response in tethered spheroids as a three-dimensional human hepatocyte in vitro model.

Cytochrome P450 (CYP) induction is a key risk factor of clinical drug-drug interactions that has to be mitigated in the early phases of drug discovery. Three-dimensional (3D) cultures of hepatocytes in vitro have recently emerged as a potentially better platform to recapitulate the in vivo liver structure and to maintain long-term hepatic functions as compared with conventional two-dimensional (2D) monolayer cultures. However, the majority of published studies on 3D hepatocyte models use rat hepatocytes and the response to CYP inducers between rodents and humans is distinct. In the present study, we constructed tethered spheroids on RGD/galactose-conjugated membranes as an in vitro 3D model using cryopreserved human hepatocytes. CYP3A4 mRNA expression in the tethered spheroids was induced to a significantly greater extent than those in the collagen sandwich cultures, indicating the transcriptional regulation was more sensitive to the CYP inducers in the 3D model. Induction of CYP1A2, CYP2B6 and CYP3A4 activities in the tethered spheroids were comparable to, if not higher than that observed in the collagen sandwich cultures. The membrane-based model is readily integrated into multi-well plates for higher-throughput drug testing applications, which might be an alternative model to screen the CYP induction potential in vitro with more physiological relevance.

HepaRG culture in tethered spheroids as an in vitro three-dimensional model for drug safety screening.

Conventional two-dimensional (2D) monolayer cultures of HepaRG cells allow in vitro maintenance of many liver-specific functions. However, cellular dedifferentiation and functional deterioration over an extended culture period in the conventional 2D HepaRG culture have hampered its applications in drug testing. To address this issue, we developed tethered spheroids of HepaRG cells on Arg-Gly-Asp (RGD) and galactose-conjugated substratum with an optimized hybrid ratio as an in vitro three-dimensional (3D) human hepatocyte model. The liver-specific gene expression level and drug metabolizing enzyme activities in HepaRG-tethered spheorids were markedly higher than those in 2D cultures throughout the culture period of 7 days. The inducibility of three major cytochrome P450 (CYP) enzymes, namely CYP1A2, CYP2B6 and CYP3A4, was improved in both mRNA and activity level in tethered spheroids. Drug-induced cytotoxic responses to model hepatotoxins (acetaminophen, chlorpromazine and ketoconazole) in tethered spheroids were comparable to 2D cultures as well as other studies in the literature. Our results suggested that the HepaRG-tethered spheroid would be an alternative in vitro model suitable for drug safety screening.